Biosensor comprising electrode for measuring hematocrit value

ABSTRACT

Provided is a method for measuring the concentration of a substance in a blood sample, comprising: supplying the blood sample to a biosensor comprising a hematocrit electrode for measuring a hematocrit value on the surface of which electrode an electrically conductive polymer is covalently immobilized; and calculating the concentration of the substance from the measured value of a first current resulting from application of a first voltage; and correcting the concentration of the substance with the value of a second current resulting from application of a second voltage or the hematocrit value calculated from the value of the second current.

TECHNICAL FIELD

The present invention relates to a biosensor comprising an electrode for measuring a hematocrit value (hereinafter, simply referred to as a hematocrit electrode).

BACKGROUND ART

In biosensors used in blood glucose self-monitoring devices, the value of the current response varies depending on the value of hematocrit in whole blood. It has been an issue that a blood glucose level deviating from its true value may be obtained as a measurement result (hematocrit influence). In view of the hematocrit influence, JP 2013-061336 A discloses a method in which the hematocrit value is measured by applying an alternating voltage in a blood glucose self-monitoring device. The measured hematocrit value is then used to correct the value of the glucose level, so that the deviation from the true value is reduced. Further, JP 4341032 B discloses a method in which a separation membrane is used to separate blood plasma from whole blood, and the blood glucose level in the plasma is then measured in order to prevent the measured glucose level from being affected by the hematocrit value.

However, the method disclosed in JP 2013-061336 A has problems in that the application of alternating voltage increases the electric power consumption by the device, and the accuracy of the blood glucose measurement may be reduced because of overcorrection due to high noise in the measurement of the hematocrit value. Further, in the method disclosed in JP 4341032 B, there are problems in that the structure of a sensor for use in the method may be complex since the sensor requires a separation membrane, and the ability of the separation membrane to separate blood cells may not be stable because of the influence of the viscosity of the sample due to components other than blood cells, such as lipids.

SUMMARY OF THE INVENTION

The present invention has been devised to solve the above-mentioned problems. Therefore, an object of the present invention is to provide a system and method by which the hematocrit value of a blood sample can be easily measured, and the concentration of a substance to be measured, such as glucose, in the sample can be accurately measured by correcting the concentration of the substance with the hematocrit value.

In order to achieve the above-mentioned object, one aspect of the present invention relates to a biosensor comprising a hematocrit electrode and an electrically conductive polymer immobilized on the surface of the hematocrit electrode. The electrically conductive polymer is preferably a water-soluble electrically conductive polymer. More preferably, the electrically conductive polymer is polyaniline. Further, the electrically conductive polymer is preferably immobilized on the surface of the electrode by a crosslinking agent.

Another aspect of the present invention relates to a biosensor comprising the above-mentioned hematocrit electrode, a working electrode, a counter electrode and a reference electrode. The working electrode is preferably an enzyme electrode. More preferably, the enzyme is an oxidoreductase.

Another aspect of the present invention relates to a method for measuring the concentration of a substance in a blood sample; comprising:

supplying the blood sample to a biosensor comprising the above-mentioned hematocrit electrode, a working electrode, a counter electrode and a reference electrode;

measuring the value of a first current which flows when a voltage of 0 to 400 mV is applied to the working electrode, and calculating the concentration of the substance from the measured value of the first current;

measuring the value of a second current which flows when a voltage of 400 to 800 mV is applied to the hematocrit electrode; and

correcting the concentration of the substance with the value of the second current or a hematocrit value calculated from the value of the second current.

Another aspect of the present invention relates to a measuring apparatus comprising:

a biosensor comprising the above-mentioned hematocrit electrode, a working electrode, a counter electrode and a reference electrode;

a control section configured to control the application of voltage to the working electrode and the hematocrit electrode of the biosensor;

a detection section configured to measure the value of a first current which flows when a voltage is applied to the working electrode, and the value of a second current which flows when a voltage is applied to the hematocrit electrode;

an arithmetic section configured to calculate the concentration of a substance from the value of the first current, and to correct the calculated concentration of the substance with the value of the second current or, alternatively, a hematocrit value calculated from the value of the second current; and

an output section configured to output the corrected concentration of the substance.

Another aspect of the present invention relates to a method for measuring a hematocrit value of a blood sample, comprising:

supplying the blood sample to a biosensor comprising a hematocrit electrode and an electrically conductive polymer immobilized on the surface of the hematocrit electrode; and

measuring the value of a current which flows when a voltage of 400 to 800 mV is applied to the hematocrit electrode.

Another aspect of the present invention relates to a measuring apparatus comprising:

a biosensor comprising a hematocrit electrode and an electrically conductive polymer immobilized on the surface of the hematocrit electrode;

a control section configured to control the application of voltage to the hematocrit electrode of the biosensor;

a detection section configured to measure the value of a current which flows when a voltage is applied to the hematocrit electrode;

an arithmetic section configured to calculate a hematocrit value from the measured value of the current; and

an output section configured to output the calculated hematocrit value.

According to the present invention, it is possible to easily and accurately measure the hematocrit value of a blood sample, and to accurately measure the concentration of a substance, such as a blood glucose level, by correcting the concentration with the hematocrit value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the structure of a biosensor according to one embodiment of the present invention.

FIG. 2 is a schematic diagram illustrating one embodiment of a measuring apparatus according to the present invention.

FIG. 3 is a flow chart illustrating one embodiment of a measurement program using the measuring apparatus according to the present invention.

FIG. 4 is a graph illustrating the results of a cyclic voltammetry analysis of blood samples with various hematocrit values, carried out using the biosensor according to one embodiment of the present invention.

FIG. 5 is a graph illustrating the relationship between the hematocrit value and the current in samples with a glucose concentration of 0 mg/ml, 100 mg/ml and 600 mg/ml, when a voltage of 600 mV was applied to a hematocrit electrode.

FIG. 6 is a graph illustrating the results of a cyclic voltammetry analysis of blood samples with various hematocrit values and with a glucose concentration of 0 mg/ml, carried out using a biosensor in which SEPLEGYDA® is used as an electrically conductive polymer.

FIG. 7 is a graph illustrating the relationship between the value of the current flowing through an enzyme electrode and the glucose concentration, in samples with various hematocrit values.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

(Hematocrit Electrode)

The hematocrit electrode according to the present invention is an electrode on the surface of which an electrically conductive polymer is immobilized.

(Electrode)

The hematocrit electrode is composed of a metallic material such as gold (Au), platinum (Pt), silver (Ag), or palladium (Pd), or a carbon material such as carbon. The hematocrit electrode is formed, for example, on an insulating base plate. The insulating base plate is composed of an insulating material, and examples thereof include various types of resins (plastics), such as thermoplastic resins (for example, polyetherimide (PEI), polyethylene terephthalate (PET) and polyethylene (PE); polyimide resins and epoxy resins; glasses; ceramics; papers; and the like. The size and the thickness of the hematocrit electrode and the insulating base plate can be selected as appropriate.

(Electrically Conductive Polymer)

Examples of the electrically conductive polymer to be immobilized on the surface of the hematocrit electrode include: polyaniline, polypyrrole, polystyrene sulfonate, polythiophene, polyisothianaphthene, polyethylenedioxythiophene(poly(3,4-ethylenedioxythiophene)poly(styrene sulfonate)), polyacrylamide sulfonate, polyvinyl sulfonate, combinations thereof, and the like. Among these, an electrically conductive polymer which can be dissolved into water to form an aqueous solution is preferred. Polyaniline is more preferred.

Examples of commercially available products of the electrically conductive polymer as described above—specifically, examples of commercially available polyaniline products include “AQUAPASS® 01-x” (manufactured by Mitsubishi Rayon Co., Ltd.). Examples of commercially available polypyrrole products include “SSPY” (ethyl 3-methyl4-pyrrolecarboxylate) (manufactured by KAKEN INDUSTRY Co., Ltd.). Examples of commercially available polystyrene sulfonate products include “Poly-NaSS” (manufactured by TOSOH ORGANIC CHEMICAL CO., LTD.). Examples of commercially available polythiophene products include “Espacer 100” (manufactured by TA Chemical Co., Ltd.). Examples of commercially available polyisothianaphthene products include “Espacer 300” (manufactured by TA Chemical Co., Ltd.). Examples of commercially available polyethylenedioxythiophene(poly(3,4-ethylenedioxythiophene)poly (styrene sulfonate)) products include “PEDOT-PSS” (Polyscience Inc.).

In an exemplary embodiment, the electrically conductive polymer is covalently attached to the surface of the electrode in order to immobilize the electrically conductive polymer on the electrode surface. To allow the electrically conductive polymer to be covalently attached to the electrode surface, for example, functional groups introduced on the electrode surface may be allowed to react with functional groups of the electrically conductive polymer to form covalent bonds. In a preferred embodiment, the electrically conductive polymer is mixed with a crosslinking agent and the polymer is covalently attached to the electrode surface via the crosslinking agent.

(Crosslinking Agent)

The crosslinking agent is not particularly limited, as long as it is capable of reacting with functional groups on the electrode surface, and also capable of reacting with the electrically conductive polymer. Examples of the crosslinking agent include aldehyde group-containing compounds, carbodiimide group-containing compounds, maleimide group-containing compounds, oxazoline group-containing compounds and epoxy group-containing compounds. Examples of aldehyde group-containing compounds as crosslinking agents include glutaraldehyde, formaldehyde, malonaldehyde, terephthalaldehyde, isobutyraldehyde, valeraldehyde, isovaleraldehyde, cinnamaldehyde, nicotinaldehyde, glyceraldehyde, glycoaldehyde, succinaldehyde, adipaldehyde, isophthalaldehyde, terephthalaldehyde, and the like. Examples of carbodiimide group-containing compounds as crosslinking agents include hexamethylene diisocyanate, hydrogenated xylylene diisocyanate, xylylene diisocyanate, 2,2,4-trimethyl hexamethylene diisocyanate, 1,12-diisocyanate dodecane, norbornane diisocyanate, 2,4-bis-(8-isocyanate octyl)-1,3-dioctyl cyclobutane, 4,4′-dicyclohexylmethane diisocyanate, tetramethyl xylylene diisocyanate, isophorone diisocyanate, and the like. The carbodiimide group-containing compounds are commercially available under the names CARBODILITE™ V-02, CARBODILITE™ V-02-L2, CARBODILITE™ V-04, CARBODILITE™ V-06, CARBODILITE™ E-02, CARBODILITE™ V-01, CARBODILITE™ V-03, CARBODILITE™ V-05, CARBODILITE™ V-07 and CARBODILITE™ V-09 (all of the above are manufactured by Nisshinbo Industries, Inc.). Examples of maleimide group-containing compounds as crosslinking agents include m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, m-maleimidobenzoyl sulfosuccinimide ester, N-γ-maleimidobutyryloxy succinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane)1-carboxylate, N-succinimidyl-2-maleimidoacetic acid, N-succinimidyl4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid, N-succinimidyl4-maleimidomethyl cyclohexane-1-carboxylic acid, N-succinimidyl4-maleimidomethyl cyclohexane-1-carboxylic acid, N-succinimidyl4-maleimidomethyl benzoic acid, N-succinimidyl-3-maleimidobenzoic acid, N-succinimidyl4-maleimidophenyl-4-butyric acid, N-succinimidyl4-maleimidophenyl4-butyric acid, N,N′-oxydimethylene-dimaleimide, N,N′-o-phenylene-dimaleimide, N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide, N-succinimidyl maleimide carboxylic acid, and the like. Examples of commercially available maleimide group-containing products include SANFEL BM-G (manufactured by SANSHIN CHEMICAL INDUSTRY Co., Ltd). Examples of oxazoline group-containing compounds as crosslinking agents include oxazoline compounds such as: 2,2′-bis-(2-oxazoline), 2,2′-methylene-bis-(2-oxazoline), 2,2′-ethylene-bis-(2-oxazoline), 2,2′-trimethylene-bis-(2-oxazoline), 2,2′-tetramethylene-bis-(2-oxazoline), 2,2′-hexamethylene-bis-(2-oxazoline), 2,2′-octamethylene-bis-(2-oxazoline), 2,2′-ethylene-bis-(4,4′-dimethyl-2-oxazoline), 2,2′-p-phenylene-bis-(2-oxazoline), 2,2′-m-phenylene-bis-(2-oxazoline), 2,2′-m-phenylene-bis-(4,4′-dimethyl-2-oxazoline), bis-(2-oxazolinylcyclohexane)sulfide, and bis-(2-oxazolinylnorbornane)sulfide. Further, examples of addition polymerizable oxazoline compounds include: 2-vinyl-2-oxazoline, 2-vinyl4-methyl-2-oxazoline, 2-vinyl-5-methyl-2-oxazoline, 2-isopropenyl-2-oxazoline, 2-isopropenyl4-methyl-2-oxazoline, 2-isopropenyl-5-ethyl-2-oxazoline, and the like. The compounds obtained by polymerization or copolymerization of one or more than one of these compounds can also be used. Examples of commercially available oxazoline group-containing compounds include Epocros WS-500, Epocros WS-700, Epocros K-1010E, Epocros K-1020E, Epocros K-1030E, Epocros K-2010E, Epocros K-2020E, Epocros K-2030E, Epocros RPS-1005 and Epocros RAS-1005 (all of the above are manufactured by NIPPON SHOKUBAI Co., Ltd.); and NK linker FX (manufactured by SHIN-NAKAMURA CHEMICAL Co., Ltd.). Specific examples of epoxy group-containing compounds as crosslinking agents include sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and the like. Two or more kinds of these compounds can be used in combination. Examples of commercially available epoxy group-containing compounds include DENACOL™ EX-611, DENACOL™ EX-612, DENACOL™ EX-614, DENACOL™ EX-614B, DENACOL™ EX-512, DENACOL™ EX-521, DENACOL™ EX-421, DENACOL™ EX-313, DENACOL™ EX-314, DENACOL™ EX-321, DENACOL™ EX-810, DENACOL™ EX-811, DENACOL™ EX-850, DENACOL™ EX-851, DENACOL™ EX-821, DENACOL™ EX-830, DENACOL™ EX-832, DENACOL™ EX-841, DENACOL™ EX-861, DENACOL™ EX-911, DENACOL™ EX-941, DENACOL™ EX-920, DENACOL™ EX-145 and DENACOL™ X-171 (all of the above are manufactured by Nagase ChemteX Corporation); SR-PG, SR-2EG, SR-8EG, SR-8EGS, SR-GLG, SR-DGE, SR-4GL, SR-4GLS and SR-SEP (all of the above are trade names, manufactured by Sakamoto Yakuhin kogyo Co., Ltd.); and Epolite 200E, Epolite 400E and Epolite 400P (all of the above manufactured by KYOEISHA CHEMICAL Co., LTD). The type of the crosslinking agent is not limited to the above-mentioned compounds and commercially available products. The crosslinking agent may also be a compound containing at least one functional group selected from an aldehyde group, a maleimide group, a carbodiimide group, an oxazoline group and an epoxy group. The form of the crosslinking agent is not limited, either and may be in the form of a monomer, a polymer or the like.

(Method for Preparing Hematocrit Electrode)

The hematocrit electrode is prepared, for example, as follows. Specifically, a metal layer which functions as an electrode is formed on one surface of the insulating base plate. For example, a metal layer having a desired thickness (for example, about 30 nm) is formed by depositing a metallic material, by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD), on one surface of the insulating base plate which is in the form of a film having a predetermined thickness (for example, about 100 μm). It is also possible to form an electrode layer composed of a carbon material, instead of a metal layer. Next, a layer of the electrically conductive polymer is formed on the electrode. Specifically, a solution (reagent) containing the electrically conductive polymer and the crosslinking agent is prepared. The solution (reagent) is dropped on the surface of the electrode. Then the solution (reagent) is allowed to dry and solidify on the electrode, to obtain the hematocrit electrode on the surface of which the electrically conductive polymer is immobilized.

It should be noted that, as with the working electrode (enzyme electrode) to be described later, an enzyme, electrically conductive particles and the like may also be immobilized on the surface of the hematocrit electrode, along with the electrically conductive polymer and the crosslinking agent. In this case, the components of the enzyme electrode and the components of the hematocrit electrode will be the same. However, this is not a problem, since the components, such as an enzyme and the like, do not influence the value of the current based on the hematocrit value, which flows when a voltage of 400 to 800 mV is applied to the hematocrit electrode. Thus, when the components of the hematocrit electrode are the same as the components of the enzyme electrode, it has the advantage of being able to produce the biosensor easily.

(Biosensor)

The biosensor according to the present invention comprises the hematocrit electrode according to the present invention.

The biosensor according to the present invention may comprise the hematocrit electrode according to the present invention alone. However, in a more preferred embodiment of the biosensor according to the present invention, the biosensor comprises a counter electrode, a working electrode (enzyme electrode), and a reference electrode, along with the hematocrit electrode according to the present invention. The counter electrode, which is used as a pair with the hematocrit electrode, may be any electrode which can be generally used as a counter electrode in a biosensor. Examples of the counter electrode include a carbon electrode prepared in the form of a film by screen printing; a metal electrode prepared in the form of a film by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD); and a silver/silver chloride electrode prepared in the form of a film by screen printing. Further, the reference electrode, which is used as a pair with the working electrode (enzyme electrode) as described herein, may also be the silver/silver chloride electrode; the carbon electrode prepared in the form of a film by screen printing; or the metal electrode prepared in the form of a film by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD).

Next, the working electrode (enzyme electrode) will be described.

(The Structure of Enzyme Electrode)

The enzyme electrode can be prepared by forming a detection layer including an enzyme, the above mentioned electrically conductive polymer, the above mentioned crosslinking agent, and electrically conductive particles as required, on an electrode.

(Enzyme Electrode)

As with the hematocrit electrode, the enzyme electrode is composed of a metallic material such as gold (Au), platinum (Pt), silver (Ag) or palladium (Pd); or a carbon material such as carbon. The electrode is formed, for example, on an insulating base plate. The insulating base plate is composed of an insulating material, and examples thereof include various types of resins (plastics), such as thermoplastic resins (for example, polyetherimide (PEI), polyethylene terephthalate (PET) and polyethylene (PE)), polyimide resins and epoxy resins; glasses; ceramics; papers; and the like. Any suitable known material can be used as an electrode material for forming the electrode and a material for forming the insulating base plate. The size and the thickness of the electrode and the insulating base plate can be selected as appropriate.

(Detection Layer)

The detection layer is in contact with the electrode and contains an enzyme, the electrically conductive polymer as described above, the crosslinking agent as described above, and may also contain other component(s) such as electrically conductive particles. However, it is preferred that the detection layer does not contain an electron mediator.

(Enzyme)

The enzyme is selected as appropriate depending on a substance to be measured, and examples thereof include oxidoreductases. Examples of oxidoreductases include glucose oxidase (GOD), galactose oxidase, bilirubin oxidase, pyruvic acid oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, L-lactic acid oxidase, ascorbic acid oxidase, cytochrome oxidase, alcohol dehydrogenase, glutamic acid dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase (GDH), fructose dehydrogenase, sorbitol dehydrogenase, lactic acid dehydrogenase, malic acid dehydrogenase, glycerol dehydrogenase, 17β hydroxysteroid dehydrogenase, estradiol 17β dehydrogenase, amino acid dehydrogenase, glyceraldehyde 3-phosphoric acid dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, cytochrome oxidoreductase, catalase, peroxidase, glutathione reductase, and the like. Among others, the enzyme is preferably a saccharide oxidoreductase. Examples of saccharide oxidoreductases include: glucose oxidase (GOD), galactose oxidase, glucose dehydrogenase (GDH), fructose dehydrogenase, and sorbitol dehydrogenase.

Further, the oxidoreductase can contain at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a catalytic subunit and a catalytic domain. Examples of oxidoreductases containing PQQ include PQQ glucose dehydrogenase (PQQGDH). Examples of oxidoreductases containing FAD include cytochrome glucose dehydrogenase (Cy-GDH) and glucose oxidase (GOD), having an α-subunit containing FAD. In addition, the oxidoreductase may contain an electron transfer subunit or an electron transfer domain. Examples of electron transfer subunits include a subunit containing heme which has a function of giving and receiving electrons. Examples of oxidoreductases having the subunit containing heme include those containing cytochrome. For example, a fusion protein of glucose dehydrogenase or PQQGDH with cytochrome can be used.

In addition, examples of enzymes containing an electron transfer domain include cholesterol oxidase and quinoheme ethanol dehydrogenase (QHEDH (PQQ Ethanol dh)). As the electron transfer domain, it is preferred to use a domain containing cytochrome containing heme which has a function of giving and receiving electrons. Examples thereof include “QHGDH” (fusion enzyme; GDH with heme domain of QHGDH)), sorbitol dehydrogenase (Sorbitol DH), D-fructose dehydrogenase (Fructose DH), glucose-3-dehydrogenase derived from Agrobacterium tumefasience (G3DH from Agrobacterium tumefasience), and cellobiose dehydrogenase. The fusion protein of PQQGDH with cytochrome, which is an example of the above mentioned subunit containing cytochrome, and a cytochrome domain of PQQGDH, which is an example of the domain containing cytochrome, are disclosed, for example, in International Publication No. WO 2005/030807. Further, as the oxidoreductase, it is preferred to use an oligomeric enzyme including at least a catalytic subunit and a subunit containing cytochrome containing heme which has a function as an electron acceptor.

(Electrically Conductive Particles)

It is preferred that the detection layer further contain electrically conductive particles. As electrically conductive particles, particles of a metal such as gold, platinum, silver or palladium; or a higher-order structure composed of a carbon material can be used. The higher-order structure may contain, for example, one or more types of fine particles (carbon fine particles) selected from particles of electrically conductive carbon black, KETJENBLACK®, carbon nanotubes (CNT) and fullerene.

Further, the surface of the detection layer may be covered with an outer-layer film composed of cellulose acetate and the like. Examples of raw materials for the outer-layer film, in addition to cellulose acetate, include: polyurethane, polycarbonate, polymethyl methacrylate, butyl methacrylate, polypropylene, polyether ether ketone, and the like.

(Method for Preparing Enzyme Electrode)

The enzyme electrode is prepared, for example, as follows. Specifically, a metal layer which functions as an electrode is formed on one surface of the insulating base plate. For example, a metal layer having a desired thickness (for example, about 30 nm) is formed by depositing a metallic material, by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD), on one surface of the insulating base plate which is in the form of a film having a predetermined thickness (for example, about 100 μm). It is also possible to form an electrode layer composed of a carbon material, instead of the metal layer.

Next, the detection layer is formed on the electrode. Specifically, a solution (reagent) containing the enzyme, the electrically conductive polymer, the crosslinking agent and the electrically conductive particles is prepared. The solution (reagent) is dropped on the surface of the electrode. Then the solution (reagent) is allowed to dry and solidify on the electrode, to obtain the enzyme electrode on the surface of which the detection layer is formed.

By using the enzyme electrode, the concentration of a substance contained in a blood sample can be measured. The substance is not particularly limited as long as it can be measured by the measuring method using the enzyme electrode. However, the substance is preferably a substance derived from a living body, which can serve as an index of a disease and/or health status, and examples thereof include glucose, cholesterol, and the like. As referred to herein a “blood sample” is a sample of untreated, i.e., whole blood, or whole blood which has been diluted or treated, but which retains red blood cells.

It is preferred that the current which is measured by the enzyme electrode is a charge transfer limiting current based on the transfer of electrons derived from the substance to the electrode. The charge transfer limiting current is a current which is generated when the electrons are transferred from the enzyme to the electrode due to the reaction between the enzyme and the substance to be measured. Further, the charge transfer limiting current is a steady-state current which does not depend on time, and preferably, a steady-state current observed after the generation of a transient current due to the charging of an electric double layer.

(Method for Producing Biosensor)

The method for producing a biosensor A according to the present invention will now be described with reference to FIG. 1. However, the following merely describes one embodiment of the present invention, and the method for producing a biosensor according to the present invention is not limited thereto.

First, four electrode patterns are printed on an insulating base plate 5, using an electrically conductive carbon ink or the like. To one of the four electrodes, a solution containing an electrically conductive polymer and a crosslinking agent is added to form a hematocrit electrode 1. Further, to another one of the four electrodes, a solution containing an enzyme, the electrically conductive polymer, the crosslinking agent, and electrically conductive particles is added to form an enzyme electrode 3. In addition, to another one of the four electrodes, a solution of silver/silver chloride is added to form a reference electrode 4. The last remaining electrode is used as it is as a counter electrode 2. Then an insulation resin is screen printed on the base plate, such that portions of these electrodes are exposed to form a reaction area 7, thereby forming an insulating layer 6.

(Method for Measuring the Concentration of Substance in Blood Sample)

The method for measuring the concentration of a substance in a blood sample according to the present invention comprises:

supplying the blood sample to the above mentioned biosensor;

measuring the value of a first current which flows when a first voltage is applied to the working electrode, and calculating the concentration of the substance from the measured value of the first current;

measuring the value of a second current which flows when a second voltage is applied to the hematocrit electrode; and

correcting the concentration of the substance with the value of the second current or a hematocrit value calculated from the value of the second current.

The application of the first voltage to the working electrode and the application of the second voltage to the hematocrit electrode may be carried out simultaneously, or may be carried out separately at different time points.

The second voltage is a constant voltage of 400 to 800 mV. The second voltage is preferably 500 to 700 mV, more preferably, 550 to 650 mV, and particularly preferably, 600 mV.

The first voltage is a constant voltage of 0 to 400 mV, and it is preferably 100 to 300 mV.

When a constant voltage of 0 to 400 mV is applied to the enzyme electrode, a current proportional to the concentration of the substance to be measured flows through the enzyme electrode, and thus, the value of the concentration (interim value of the concentration) of the substance, such as glucose, can be obtained from the value of the current. The measurement of the current which flows through the enzyme electrode is preferably carried out 3 to 60 seconds after the application of voltage.

Further, if the concentration of the substance, such as glucose, is constant, the current which flows through the hematocrit electrode when a constant voltage of 400 to 800 mV is applied thereto will be negatively correlated with the hematocrit value of the sample. The measurement of the current which flows through the hematocrit electrode is preferably carried out 3 to 60 seconds after the application of voltage. If the relationship between the value of the current which flows through the hematocrit electrode when a constant voltage of 400 to 800 mV is applied, and the hematocrit value, at respective concentrations of the substance, is obtained in advance, the hematocrit value of the blood sample used in the measurement can be obtained from the above mentioned interim value of the concentration and the value of the current which flows through the hematocrit electrode. This may be achieved, for example, by reference to calibration curves or use of a formula for the calculation.

Further, if the relationship between the concentration of the substance and the value of the current which flows through the enzyme electrode, and the influence of the hematocrit value thereon, is obtained in advance, it is possible to accurately measure the concentration of the substance, such as glucose, by correcting the value of the concentration with the hematocrit value. This may be achieved, for example, by reference to calibration curves or use of a formula for the calculation, as described in relation to the measuring apparatus described hereinafter. When the relationship between the value of the current which flows through the hematocrit electrode and the hematocrit value is known in advance, the value of the concentration of the substance may be corrected with the value of the current which flows through the hematocrit electrode, without calculating the hematocrit value.

(Method for Measuring Hematocrit Value of Blood Sample)

The method for measuring the hematocrit value of a blood sample according to the present invention comprises:

supplying the blood sample to a biosensor comprising a hematocrit electrode and an electrically conductive polymer immobilized on the surface of the hematocrit electrode; and

measuring the value of a current which flows when a voltage of 400 to 800 mV is applied to the hematocrit electrode.

If the relationship between the value of the current which flows through the hematocrit electrode when a constant voltage of 400 to 800 mV is applied, and the hematocrit value, at respective concentrations of the substance to be measured, is obtained in advance, the hematocrit value of the blood sample used in the measurement can be obtained from the above mentioned interim value of the concentration and the value of the current which flows through the hematocrit electrode.

(Measuring Apparatus for Measuring Substance to be Measured in Blood Sample)

A measuring apparatus B according to the present invention will be described with reference to the drawings. Although a glucose measuring apparatus which includes a glucose sensor as the biosensor is illustrated in this embodiment, the measuring apparatus according to the present invention is not limited to the following embodiment.

The measuring apparatus according to the present invention comprises:

the above mentioned biosensor;

a control section configured to control the application of voltage to the working electrode and the hematocrit electrode of the biosensor;

a detection section configured to measure the value of a first current which flows when a first voltage is applied to the working electrode, and the value of a second current which flows when a second voltage is applied to the hematocrit electrode;

an arithmetic section configured to calculate the concentration of a substance from the value of the first current, and to correct the concentration of the substance with the value of the second current or a hematocrit value calculated from the value of the second current; and,

-   -   an output section configured to output the corrected         concentration of the substance.

FIG. 2 shows an example of the configuration of main electronic components included in the measuring apparatus B. A control computer 18, a potentiostat 19 and a power supply device 11 are provided on a base plate 20 housed in a housing. The control computer 18 includes, as hardware, a processor such as CPU (central processing unit); a recording medium such as a memory (RAM (Random Access Memory) or ROM (Read Only Memory)); and a communication unit. When the processor loads a program stored in the recording medium (for example, the ROM) to the RAM, and executes the program, the control computer 18 functions as an apparatus including an output section 10, a control section 12, an arithmetic section 13 and a detection section 14. The control computer 18 may also include an auxiliary memory such as a semiconductor memory (EEPROM or flash memory) or a hard disk.

The control section 12 controls the timing for applying the voltage and the value of the voltage to be applied. The power supply device 11 includes a battery 16, and supplies electricity to the control computer 18 and the potentiostat 19 for operation. It is also possible to dispose the power supply device 11 outside the housing. The potentiostat 19 is a device which maintains the potential of the working electrode constant with respect to the potential of the reference electrode.

The potentiostat 19, which is controlled by the control section 12, applies a predetermined amount of voltage between the counter electrode (reference electrode) and the working electrode of a glucose electrode (enzyme electrode) 17, and between the counter electrode and the working electrode of a hematocrit electrode 21, using terminals CRs and Ws; measures the response currents of the working electrodes which can be obtained at the terminals Ws, respectively, and send the results of the measurement to the detection section 14. The reference herein to a “glucose electrode” is an enzyme electrode in which the enzyme reacts with glucose to generate a current by transfer of electrons from the enzyme to the electrode.

The description which follows, refers to glucose as the substrate to be measured for illustration purposes. The arithmetic section 13 calculates the glucose concentration from the value of the current measured at the glucose electrode, and calculates the hematocrit value (this step can be omitted when the glucose concentration is to be corrected with the value of the current which flows through the hematocrit electrode) from the value of the current measured at the hematocrit electrode, respectively; corrects the glucose concentration with the value of the second current or the hematocrit value calculated based on the value of the second current in the arithmetic section; and stores the obtained value. The output section 10 carries out data communication between the output section 10 and a display section unit 15, and sends the calculated result of the concentration of the substance provided by the arithmetic section 13 to the display section unit 15. The display section unit 15 is capable of displaying, for example, the calculated result of the glucose concentration which is received from the measuring apparatus B, on a display screen in a predetermined format.

FIG. 3 is a flow chart showing an example of the processing sequence of the glucose concentration measurement carried out by the control computer 18. When the CPU (control section 12) of the control computer 18 receives an instruction to start the measurement of the glucose concentration, the control section 12 controls the potentiostat 19 to apply the predetermined amount of voltage to each of the glucose electrode and the hematocrit electrode, and starts measuring each of the response currents (Step S01). The detection of the installation of a sensor to the measuring apparatus may be used as the instruction to start the measurement of the concentration.

Next, the potentiostat 19 measures the response current obtained by the application of voltage, specifically, the response current at the glucose electrode and the response current at the hematocrit electrode, and sends the measured current values to the detection section 14 (Step S02).

The arithmetic section 13 carries out arithmetic processing based on the current values, and calculates the glucose concentration and the hematocrit value (calculation of the hematocrit value can be omitted when the glucose concentration is to be corrected with the value of the current which flows through the hematocrit electrode) (Step S03). For example, the formulae for calculating the glucose concentration or the data of the calibration curve of the glucose concentration are preinstalled to the arithmetic section 13 in the control computer 18, and the arithmetic section 13 calculates the (interim) glucose concentration utilizing these calculation formulae or the calibration curve.

Further, the formulae or the data of the calibration curve representing the relationship between the current value and the hematocrit value at respective glucose concentrations are also preinstalled to the arithmetic section 13 in the control computer 18, and the arithmetic section 13 calculates the hematocrit value using these formulae or the data of the calibration curve and the calculated (interim) glucose concentration.

In addition, the arithmetic section 13 corrects the (interim) glucose concentration with the hematocrit value, to calculate the corrected glucose level (Step S04). For example, the formulae or the data of the calibration curve representing the relationship between the hematocrit value and the glucose concentration are preinstalled to the arithmetic section 13 in the control computer 18, and the arithmetic section 13 corrects the glucose concentration using these formulae or the data of the calibration curve.

The output section 10 sends the calculated result of the corrected glucose concentration to the display section unit 15, through a communication link provided between the output section 10 and the display section unit 15 (Step S05). Thereafter, the control section 12 determines if there are any measurement errors detected (Step S06); completes the measurement if there is no error; and displays the glucose concentration on the display section. If there are any errors, a notification of error is displayed, and then the flow sequence shown in FIG. 3 is completed.

(Apparatus for Measuring Hematocrit Value)

The apparatus for measuring a hematocrit value according to the present invention comprises:

a biosensor comprising a hematocrit electrode and an electrically conductive polymer immobilized on the surface of the hematocrit electrode;

a control section configured to control the application of voltage to the hematocrit electrode of the biosensor;

a detection section configured to measure the value of a current which flows when a voltage is applied to the hematocrit electrode;

an arithmetic section configured to calculate a hematocrit value from the measured value of the current; and an output section configured to output the calculated hematocrit value.

EXAMPLES

Examples of the biosensor will now be described. However, the following Examples are merely illustrative of one embodiment of the present invention, and the content of the present invention is not limited by the following embodiment.

<Preparation of Electrodes>

As shown in FIG. 1, patterning was performed on one surface of a polyethylene terephthalate base material (E-22; manufactured by Toray Industries, Inc.) (length: 50 mm, width: 5 mm, thickness: 250 μm) by a screen printing method, using an electrically conductive carbon ink as a base electrode material, to form a four-electrode pattern. One of the four electrodes was left as it is to be used as a counter electrode. On the surface of another one of the four electrodes, a silver/silver chloride ink (manufactured by BAS Inc.) was coated, and the resulting base material was dried at 80° C. for 20 minutes to form a silver/silver chloride electrode, which was used as a reference electrode. Further, a reagent solution to be described later was coated on each of the remaining two electrodes, and the resulting base material was dried at 100° C. in a drying furnace for two hours to prepare a hematocrit electrode and an enzyme electrode (working electrode) respectively.

After forming the four-electrode pattern by patterning and before coating the reagent to each of the electrodes, an insulation resin polyester ink (UVF series; manufactured by Asahi Chemical Research Laboratory Co., Ltd.) was printed on top of the electrodes by screen printing to form an insulating layer, such that the insulating layer has a window which allows one portion of each of the electrodes to be exposed. Finally, a hydrophilic film, which had its thickness restricted by a double-sided adhesive tape, was pasted on top of the resulting base material to form a capillary, thereby obtaining the biosensor.

(Reagent Solution)

A reagent solution as described below was prepared, and used for the above mentioned hematocrit electrode and the enzyme electrode (working electrode).

KETJENBLACK® (manufactured by Mitsubishi Carbon Black): 1.20%

Electrically conductive polymer: aqueous sulfonated polyaniline solution (AQUAPASS®-01x, manufactured by Mitsubishi Rayon Co., Ltd.): 0.40%

Oxazoline group-containing polymer (EPOCROS® WS-700, manufactured by NIPPON SHOKUBAI Co., Ltd.): 6.0%

Enzyme (Cy-GDH): 4.5 mg/mL

Phosphate buffer solution (pH 5.8): 10 mM

Sucrose: 0.5%

Here, “%” represents the percent by weight concentration of the reagent contained in the reagent solution.

(Cyclic Voltammetry)

The response characteristics of the hematocrit electrode of the biosensor were evaluated by analyzing the cyclic voltammetry waveforms. The cyclic voltammetry waveforms were obtained as follows: whole blood samples having a glucose concentration of 0 mg/dL and a hematocrit value of 20%, 42% and 70% were each introduced into a sample supplying portion (reaction area) of the biosensor, then the voltage applied to the hematocrit electrode was swept from −800 mV→+800 mV, at a sweep rate of 20 mV/sec, and the response current during the sweeping was measured. FIG. 4 shows the cyclic voltammetry waveforms obtained by the measurement. It can be seen from FIG. 4 that, when the applied voltage is around 600 mV (from 400 to 800 mV), the value of the current varies depending on the hematocrit value. The reason for this is thought to be due to the influence of the shielding effect by the blood cells, and the reaction between component(s) in blood plasma and the electrically conductive polymer.

Further, when the same experiment was carried out using blood samples having a different glucose concentration, it was found that, also in samples having a glucose concentration of 100 mg/dL and of 600 mg/dL, the value of the current varies depending on the hematocrit value, when the applied voltage is around 600 mV (400 to 800 mV).

FIG. 5 illustrates the relationship between the current value and the hematocrit value in samples having different glucose concentrations, when a voltage of 600 mV was applied to the hematocrit electrode. It can be seen from FIG. 5 that the current value is negatively correlated with the hematocrit value, when the applied voltage is around 600 mV (400 to 800 mV).

When the measurement was performed using a biosensor in which the hematocrit electrode contains no electrically conductive polymer, the value of the current when a voltage of around 600 mV (400 to 800 mV) was applied, did not vary depending on the hematocrit value (not shown).

Likewise, when the measurement was performed with a biosensor in which SEPLEGYDA® (manufactured by Shin-Etsu Polymer Co., Ltd.) is used instead of AQUAPASS®-01x, as the electrically conductive polymer, as well, it was found that the value of the current when a voltage of 600 mV was applied to the electrode is negatively correlated with the hematocrit value (FIG. 6).

FIG. 7 illustrates the relationship between the value of the current flowing through the enzyme electrode and the glucose concentration in samples with various hematocrit values, obtained by a separate measurement. The hematocrit value can be calculated from the value of the current flowing through the hematocrit electrode when a voltage of 600 mV was applied thereto; and from the thus obtained hematocrit value, and the value of the current flowing through the enzyme electrode when a voltage of 0 to 400 mV was applied thereto, a glucose concentration corrected with the hematocrit value can be calculated.

DESCRIPTION OF SYMBOLS

-   A biosensor -   1 hematocrit electrode -   2 counter electrode -   3 enzyme electrode -   4 reference electrode -   5 base plate -   6 insulating layer -   7 reaction area -   B measuring apparatus -   10 output section -   11 power supply device -   12 control section -   13 arithmetic section -   14 detection section -   15 display section unit -   16 battery -   17 glucose sensor -   18 control computer -   19 potentiostat -   20 base plate -   21 hematocrit electrode -   CR, W terminals     While the invention has been described in detail with reference to     preferred embodiments thereof, it will be apparent to one skilled in     the art that various changes can be made, and equivalents employed,     without departing from the scope of the invention. Each of the     aforementioned documents as well as JP2015-077650 is incorporated by     reference herein in its entirety. 

What is claimed is:
 1. A biosensor comprising: an enzyme electrode configured to measure a glucose concentration in a blood sample, wherein a surface of the enzyme electrode is in contact with a detection layer comprising an oxidoreductase enzyme, a first electrically conductive polymer and electrically conductive particles; and a hematocrit electrode configured to measure a hematocrit value in the blood sample, wherein the hematocrit electrode comprises a metal layer or a carbon layer formed on an insulating base plate and wherein a second electrically conductive polymer is covalently immobilized on a surface of the metal layer or the carbon layer by a crosslinking agent.
 2. The biosensor according to claim 1, wherein the first and second electrically conductive polymers are water-soluble electrically conductive polymers.
 3. The biosensor according to claim 1, wherein the first and second electrically conductive polymers are a polyaniline.
 4. The biosensor according to claim 1, wherein the crosslinking agent contains at least one functional group selected from the group consisting of an aldehyde group, a maleimide group, a carbodiimide group, an oxazoline group, an epoxy group and combinations thereof.
 5. The biosensor according to claim 1, further comprising a counter electrode and a reference electrode.
 6. The biosensor according to claim 1, wherein the detection layer of the enzyme electrode further comprises a crosslinking agent.
 7. The biosensor according to claim 1, wherein the oxidoreductase is saccharide oxidoreductase.
 8. A method for reducing deviation in the measured concentration of blood glucose as an index of a disease or health status, comprising: contacting a blood sample containing glucose with the detection layer of the enzyme electrode of the biosensor according to claim 1; reacting the glucose in the blood sample with the oxidoreductase present in the detection layer by applying a first voltage of 100 to 300 mV to the enzyme electrode; measuring the value of a resulting first current which flows from the first voltage, and calculating the concentration of the glucose from the measured value of the first current; applying a second voltage of 400 to 800 mV to the hematocrit electrode; measuring the value of a resulting second current which flows from the second voltage and calculating a hematocrit value from the measured value of the second current; and correcting the measured glucose concentration to account for the hematocrit value.
 9. The method according to claim 8, wherein the second voltage is 550 to 650 mV.
 10. A measuring apparatus comprising: the biosensor according to claim 1; a control section configured to control the application of voltage to the enzyme electrode and the hematocrit electrode of the biosensor; a detection section configured to measure the value of a first current which flows when a voltage is applied to the enzyme electrode, and the value of a second current which flows when a voltage is applied to the hematocrit electrode; an arithmetic section configured to calculate the concentration of blood glucose from the value of the first current, and to correct the concentration of the blood glucose with the value of the second current or the hematocrit value calculated from the value of the second current; and an output section configured to output the corrected concentration of the blood glucose to be measured. 